Cellular specificity of plasmacytoma-induced immunosuppression.

The pattern of immunodeficiency in plasmacytoma-bearing mice appears to be unique. Mice bearing these tumors exhibit a severe impairment in their ability to mount a primary immune response to thymus-dependent and -independent antigens. However, cell-mediated immune functions in these mice apparently remain intact. Thus, when T cell activity of lymph node cells from plasmacytoma-bearing mice was tested in vivo by sensitization with dinitrofluorobenzene and in vitro by responsiveness to phytohemmagglutinin, allogeneic cells, and dinitrobenzene sulfonate, cell-mediated immunity was found to be normal.     

Double immunocytochemical staining for in vivo detection of epitope specificity and isotype of antibody-forming cells against synthetic peptides homologous to human immunodeficiency virus-1.

Many infections evoke a strong humoral immune response. Some (e.g., HIV-1, EBV, CMV) also lead to disorders of the B-cell system. Data concerning cell dysfunction are largely derived from in vitro studies, which necessarily exclude all microenvironmental influences. The aim of this study was to develop a tool for the investigation of epitope specific humoral immune responses in vivo. Mice were immunized with one of two synthetic peptides, both 21 amino acids long and homologous to regions of the HIV-1 gp160. Cryostat sections of spleen and lymph nodes were incubated with the corresponding peptide coupled to alkaline phosphatase and simultaneously incubated with peroxidase-conjugated rabbit antisera specific for mouse immunoglobulin isotypes. We were able to show simultaneous detection of epitope specificity, isotype, and localization of antibody-forming cells and immune complexes in tissue sections. It should prove useful for in vivo investigation of the development of specific (e.g., anti-HIV-1) humoral immune response, the determination of B-cell specificity in lymph node infiltrates, and the role of immune complexes in lymph node pathology.     

An electron microscope autoradiographic study of DOC conversion to corticosterone in the rat adrenal cortex

Summary Seven thymuses from children between 1 and 12 years were examined by electron microscopy. Biopsies had been taken during surgical correction of congenital heart defects.In all cases we found interdigitating reticulum cells (IRC) in the medulla and inner cortex. These cells resembled the IRC which have been described previously in the thymus-dependent regions of the spleen and lymph node. They were characterized by an irregularly shaped nucleus, narrow cisterns of rough endoplasmic reticulum, and widespread interdigitation and invagination of the cell membrane. The surfaces of the IRC were in close contact with those of small lymphocytes, sometimes polysomal lymphatic cells, epithelial cells, and occasionally with those of lymphatic cells containing ergastoplasm.The IRC is apparently a specific cell of thymus-dependent regions. It may be that the IRC in the thymus, lymph node, and spleen contribute to the microenvironment needed for the differentiation of T-cells.Supported by the Deutsche Forschungsgemeinschaft, SFB 111/CII and III.—We wish to thank Miss M. Neubert and Mrs. R. Köpke for their technical assistance and Mrs. M. Soehring for her help with the translation.     

Cell-mediated and humoral immune responses to aspermatogenic antigen in experimental allergic orchitis in the guinea-pig

Guinea-pigs were immunized with a defined and highly potent aspermatogenic antigen, G75m, and the occurrence of orchitis was correlated with (1) cell-mediated immune response to G75m, determined by lymph node cell proliferation and by secretion of macrophage migration inhibitory factor (MIF) by peritoneal exudate cells, and (2) humoral antibodies to G75m and to cell surface antigens of guinea-pig testicular cells, by radioimmunometric assays. A consistent temporal relationship between cell-mediated immune responses and disease was found: lymph node cell proliferation was positive by Day 4, followed 3 days later by maximum secretion of MIF, and orchitis lesions were manifest on Day 10. In contrast, maximal IgG antibodies to G75m or to the surface antigens of spermatozoa/testicular cells were detected at a time when cell-mediated immune responses and active testicular lesions had subsided. In individual animals, lymph node cell proliferation increased with severity of orchitis, while MIF secretion by peritoneal cells increased with orchitis only late in the disease. Early in disease, MIF response showed a negative correlation with orchitis. Moreover, peritoneal injection of oil reduced the incidence of early lymph node cell proliferative responses, and delayed the onset of testicular disease. These findings are consistent with competition between different inflammatory sites for recently antigen-activated T lymphocytes. We conclude that (1) the development of orchitis correlates with cell-mediated immune responses to purified aspermatogenic antigens but not with IgG antibody responses, and (2) when the same animal is used to assess different aspects of cellular immunity and autoimmune disease, one study may significantly influence the other.     

Thymus dependency of cells involved in transfer of delayed hypersensitivity to listeria monocytogenes in mice

Delayed-type hypersensitivity (DH) to Listeria antigens was induced in inbred C3Hf/Umc mice by intravenous injection of a sublethal dose of viable Listeria monocytogenes. Bone marrow, spleen, and lymph node cells from the immune mice were capable of passive transfer of DH to syngeneic neonatally thymectomized or lethally (900 R) irradiated recipients. Immune thymus cells as well as immune serum were ineffective in transferring DH to irradiated animals. In vitro treatment with antitheta isoantibody (anti-θ) and complement abolished the capacity of spleen and bone marrow cells from immune donors to transfer DH to irradiated hosts, indicating the thymus dependency of this cell population. The results with bone marrow indicate the existence of a small, but biologically significant, thymus-dependent population in this tissue.     

Correlation of cytotoxicity with experimental allergic encephalomyelitis.

Cytotoxicity of immune lymph node cells in experimental allergic encephalomyelitis (EAE) was maximal 9 days after injection of encephalitogenic emulsion. The ability of these cells to passively transfer EAE was also maximal at this time. Immune spleen cells were more cytotoxic than lymph node cells 9 days after injection; however, these cells did not passively transfer EAE. Twelve days after injection of encephalitogenic emulsion immune spleen cells passively transferred EAE with resulting mild histopathologic lesions. At this time the spleen cells were 50% more cytotoxic than comparable lymph node cells. Cyclophosphamide suppressed the development of clinical EAE and the development of cytotoxic lymphoid cells. It also reduced clinical signs and cytotoxic activity of lymph node cells. Spleen cell cytotoxic activity was enhanced by Cyclophosphamide. It was concluded that cytotoxic activity of lymph node and spleen cells was correlated with the ability of these cells to produce EAE. Lymph node cell populations differed qualitatively and/or quantitatively from immune spleen cell populations in EAE. Capacity to passively transfer EAE coincided with the maximal Cytotoxicity of the lymphoid cells from each tissue.     

The early postnatal development of the primary immune response in rat popliteal lymph node,stimulated with thymus-independent type-1 and type-2 antigens

Summary To examine the development of the postnatal immune response to thymus-independent type-1 (TI-type 1) and TI type-2 antigens, respectively, trinitrophenyl-lipopolysaccharide (TNP-LPS) or TNP-Ficoll was injected subcutaneously into the hind footpads of young rats of various ages. After 5 days the popliteal lymph nodes (PLNs) were removed and the localization pattern of specific anti-TNP antibody-containing cells was studied. The first specific antibody-containing cells elicited in rats by TNP-LPS appeared in animals at day 19 after birth. The results suggest that the development of these cells from lymphocyte to plasma cell occurs while they migrate from cortex to medulla. An unexpected finding was the low response to TNP-Ficoll in PLN; from 6 weeks after birth only very few specific antibody-containing cells were found. However, in the spleen numerous anti-TNP antibody-containing cells were found in the periarteriolar lymphocyte sheaths. To test the exclusive role of the spleen in the appearance of anti-TNP antibody-containing cells in lymph node after subcutaneous administration of TNP-Ficoll, the experiment was repeated in rats that had been splenectomized. Evidence from these experiments suggests that the spleen plays a major role in the appearance of the above-mentioned cells in lymph nodes.Abbreviations KLH keyhole-limpet haemocyanin - LPS lipopolysaccharide - PBS phosphate-buffered saline - PLN popliteal lymph node - TD thymus-dependent - TI thymus-independent - TNP trinitrophenyl     

Regulation of parasite antigen-induced T cell growth factor activity and proliferative responsiveness in Brugia pahangi-infected jirds

Previous studies have demonstrated that the induction of immunoregulatory mechanisms in the spleens of Brugia pahangi-infected jirds is correlated with the onset of microfilaremia. This study investigated the relationship between production of a factor with IL-2-like activity and the regulation of T cell-mediated responses in jirds experimentally infected with B. pahangi. A factor present in culture supernatants of mitogen-stimulated jird lymphocytes supported the proliferation of murine CTLL cells and provided the basis for an IL-2 assay. Mitogen induced proliferative responses and IL-2 production of spleen cells but not lymph node cells from pre-patent and microfilaremic jirds were suppressed. Both B. pahangi Ag-induced proliferative responsiveness and IL-2 production of spleen cells from microfilaremic jirds were also suppressed relative to lymph node cells from the same animals or spleen cells from B. pahangi immunized or prepatent jirds. Depletion of histamine receptor-bearing cells restored the ability of spleen cells from microfilaremic jirds to produce significant levels of IL-2. In addition, in add-mixture experiments, spleen cells from microfilaremic jirds suppressed Ag-induced IL-2 production by cells from either B. pahangi- or KHL-immunized jirds. Exogenous IL-2 failed to reconstitute the suppressed Ag-induced proliferative response of spleen cells from microfilaremic jirds. This study demonstrates that the down-regulation of immune responses in B. pahangi infection is a cell-mediated event and is associated with an inability to produce IL-2.     

In vitro murine lymphocyte blastogenic responses to Cryptosporidium parvum.

Spleen and mesenteric lymph node lymphocytes from both Cryptosporidium parvum-exposed and unexposed mice were cultured with antigen (Ag) prepared from C. parvum oocysts. Spleen lymphocytes from oral-, intraperitoneal-, or oral + intraperitoneal-exposed mice did not respond significantly (P greater than 0.05) to Ag stimulation. Spleen lymphocytes from multioral-exposed mice, however, demonstrated significant (P less than or equal to 0.01) Ag-specific blastogenesis. Mesenteric lymph node lymphocytes did not respond to in vitro Ag stimulation regardless of the route of in vivo priming. These results demonstrate an in vitro cell-mediated immune response against C. parvum by lymphocytes in murine spleen.     

Lymphocyte migration during the development of regional lymph node anergy in experimental tumor growth

The development of lymph node anergy in Wistar rats to growing Walker carcinoma 256 was studied in vitro using the 51Cr-release cytotoxicity assay. Cell-mediated cytotoxicity to the tumor peaked in draining lymph nodes 11 days after tumor transplantation. By 14 days, the regional lymph node had become anergic to the tumor at a time when cell-mediated cytotoxicity was still increasing in the more distal contralateral lymph node. Lymphocyte migration into resting, cytotoxic, and anergic lymph nodes was analyzed to determine if altered cell migration into the regional lymph node was associated with the development of anergy. Lymphocyte migration was found to be enhanced in both cytotoxic and anergic regional lymph nodes of tumor-bearing animals. It is concluded that lymph node anergy in this experimental tumor system is not related to changes in lymphocyte migration patterns; rather, it is the result of alterations in the microenvironment of the lymph node which prevents the expression of cytotoxic effector cells.     

Contact sensitivity to picryl chloride: the occurrence of B suppressor cells in the lymph nodes and spleen of immunized mice.

Mice were immunized for contact sensitivity and antibody production by painting the skin with picryl chloride. Lymph node and spleen cells taken 4 days later transferred contact sensitivity. However, cells taken at 7–8 days failed to transfer but were able to block the transfer by 4 day immune cells. These suppressor cells occurred in the regional lymph nodes, spleen and thymus. The suppressor activity of lymph node and spleen cells was due to B cells as shown by the effect of anti-θ serum and complement, nylon wool filtration and separation of EAC positive and negative cells by centrifugation on a discontinuous gradient. The transfer of fractions rich or poor in macrophages showed that the suppressor cell in the transferred population was not a macrophage. Separation using EAC rosettes suggested that B cells were responsible for the suppressor activity in the thymus.T cells isolated from the lymph nodes and spleen 7–8 days after immunization transferred contact sensitivity although the initial population was inactive. This indicates that passive transfer cells are present in the regional lymph nodes and spleen at later times after immunization but cannot be demonstrated because of the presence of suppressor B cells. However, no passive transfer cells were found in the thymus. The production of B suppressor cells required little or no T cell help and following immunization the spleens of reconstituted (B) mice were at least as active as control cells in causing suppression. There are several different suppressor cells which act in the picryl system and the B suppressor cells in immunized mice described here are distinct from the T suppressor cells in mice injected with picryl sulphonic acid.     

Adherence of germ tubes of Candida albicans to tissues from immunocompromised mice

Abstract The influence of immune status of the host on binding of germ tubes of Candida albicans to murine tissue sections in an ex vivo assay was examined. Generally, germ tubes appeared randomly adhered to the tissues examined and binding was unaffected by immunodeficiency induced by treatment with cyclophosphamide and cortisone acetate. Adherence was somewhat reduced in spleen and kidney sections or increased in liver sections and unchanged in lymph node sections from treated mice compared to sections from control animals. Scanning electron micrographs showed organisms appeared to be loosely or tightly bound to the surface or partially embedded in spleen sections from both control and treated mice. These observations suggested that qualitative and quantitative difference in adhesion of germ tubes to various tissues may contribute little to the susceptibility of the immunodeficient animal to candidal infection.     

Regulation of cyclophosphamide-induced eosinophilia in contact sensitivity: functional roles of interleukin-5-producing CD4(+) lymphocytes

Blood and tissue eosinophilia is obtained when mice pretreated with cyclophosphamide (CY) and sensitized with picryl chloride are challenged on each ear lobe on day 13. To gain important insights into the cellular mechanisms involved in CY-induced eosinophilia in the contact-sensitivity reaction, we examined the cytokine profile expressed in regional lymph node cells and spleen cells. CY pretreatment 2 days before sensitization enhanced expression of IL-4 mRNA in the regional lymph node cells more strongly than expressions of both IL-2 and IFN-gamma mRNA on day 13. Five days after sensitization, spleen cells expressed IL-5 mRNA and produced IL-5 in vitro. Depletion of CD4(+) cells from spleen cells completely abrogated the secretory capacity of IL-5. In vivo blocking of IL-5 on day 3 entirely inhibited spleen, bone marrow, and subsequent blood eosinophilia. When immune lymph node cells prepared on day 13 were stimulated with hapten-modified cells in vitro, the level of IL-4 secreted in the culture supernatant was enhanced by CY pretreatment, but that of IL-2 was not. One important result was that IL-5 was not produced in response to in vitro stimulation, despite the fact that marked eosinophil infiltration in the dermis was observed in vivo. Thus, eosinophilopoiesis was stimulated by IL-5-producing CD4(+) immune T cells that were present in the eosinophil production site, particularly in the spleen before elicitation. In contrast, eosinophil recruitment into the dermis in the efferent phase can be induced without production of IL-5 from lymphocytes.     

The antimetastatic function of concomitant antitumor immunity. II. Evidence that the generation of Ly-1+2+ effector T cells temporarily causes the destruction of already disseminated tumor cells

Progressive growth of the P815 mastocytoma in an immunocompetent host evokes the generation of an antitumor immune response that can be measured in terms of the production of cytolytic Ly-1+2+ T cells in the draining lymph node and spleen. This immunity, designated concomitant immunity, is present on day 6 of tumor growth, peaks on day 9, and decays progressively thereafter. It fails to develop in mice made T cell deficient by thymectomy and lethal whole-body gamma-radiation, and reconstituted with syngeneic bone marrow cells (TXB mice). Employment of a mouse survival assay, capable of enumerating metastatic P815 cells in cell suspensions, showed that the P815 tumor metastasizes to the draining lymph node and spleen at the same rate in normal and TXB mice for the first 6 days of growth of an intradermal P815 tumor. By day 6 of tumor growth there were approximately 10(3) P815 cells in the draining lymph node in both types of mice. However, during the generation of concomitant immunity between days 6 and 9, the number of metastatic P815 cells in the draining lymph nodes and spleens of normal tumor-bearing mice declined by nearly 90%. After day 12, however, the number of tumor cells in the nodes and spleens increased concordantly with the decay of concomitant immunity. These findings, together with the demonstration that T cell-deficient mice failed to restrain the number of metastatic P815 cells in the draining lymph node and spleen, suggest that concomitant immunity is an important defense mechanism against the development of systemic disease. Additional evidence consistent with this interpretation was provided by studies which showed that adoptive immunization with spleen cells from concomitant immune donors significantly prolonged the median survival time of TXB tumor-bearing mice by destroying a substantial proportion of P815 tumor cells already seeded in the draining lymph node. Adoptive immunization also delayed the appearance of metastatic tumor cells in the spleen.     

Cellular interactions in the generation and expression of histamine-induced suppressor activity

The tissue, anatomic, and developmental distribution of Maclura pomifera (MP) lectin binding to rat lymphoid cells was examined. Analysis was performed by immunofluorescence microscopy and by the fluorescence-activated cell sorter. Comparison with anti-rat Ig to detect B cells and the monoclonal antibodies W3/13, W3/25, and OX 8 to detect T cells revealed that the MP lectin reacted with all T cells and not B cells in spleen and lymph node of young adult rats. The lectin also bound selectively to the thymus-dependent areas in frozen sections of spleen and lymph node. Using the MP lectin in conjunction with anti-Thy1 antibody and the monoclonal antibodies, W3/25 and OX 8, four T-cell subpopulations in spleen and lymphnode were identified on the basis of their cell surface antigenic phenotype. The T-cell developmental distribution of MP binding revealed that 100% of normal and neoplastic thymocytes bound the lectin whereas approximately 25% of TdT⁺ bone marrow cells, putative thymocyte progenitors, were MP⁺. Thus, the MP lectin is a nonimmunoglobulin reagent which binds to prethymic, thymic, and post-thymic cells of the T-lymphocyte lineage. Affinity chromatography experiments indicated that the MP lectin binds, at least in part, to the major thymocyte cell surface glycoprotein which is recognized by the W3/13 monoclonal antibody.     

Altered lymphocyte functions in rats bearing syngeneic Moloney sarcoma tumors. I. Mitogen responses, mixed lymphocyte reactions (MLR) and mixed lymphocyte-tumor reactions (MLTR).

Impairment of mitogen responses to Con A and LPS and of MLR and MLTR was detected in the spleens of rats bearing syngeneic Moloney sarcoma tumors. Depressed responses of both T cell and Ig+ cell populations were observed. During the observation period of 6 to 10 days post-tumor inoculation when maximal T cell-mediated cytotoxicity was observed in spleen and draining lymph node cells, spleen cells showed marked impairment in response to stimuli mentioned above. By contrast, draining lymph node cell activity was either unaltered or somewhat elevated above the level of activity measured in normal control populations. Data presented in this and an accompanying paper strongly indicate that macrophages are activated as immunosuppressor cells in tumor-bearing rats.     

Characteristics of the immunosuppressive activity of lymphoid organ cells in the process of sensitization with ram erythrocytes and exposure to antilymphocyte serum

In the adoptive transfer of cells obtained from the thymus, lymph nodes and the spleen to intact syngeneic animals the suppression of immune response was induced by lymph node cells. If the donors were previously sensitized, the cells of the thymus and lymph nodes showed suppressive activity in the adoptive transfer test. A single injection of antilymphocytic serum to the donors of lymphoid cells, previously sensitized with sheep red blood cells, enhanced the immunosuppressing action of thymocytes and lymph node cells.     

Inhibition of specific immune responses by feeding protein antigens. IV. Evidence for tolerance and specific active suppression of cell-mediated immune responses to ovalbumin.

We studied the effect of a single intragastric administration of ovalbumin (OVA) on the subsequent development of OVA-specific cell-mediated immune (CMI) responses in BDF1 mice. In animals fed OVA 7 days before subcutaneous sensitization with OVA-CFA, we observed a concomitant dose-dependent decrease in both the humoral and CMI responses specific for OVA. The CMI tolerance was found to be antigen-specific when assayed in vivo by ear swelling or in vitro by an antigen-induced T cell proliferation assay because OVA-fed mice responded normally to sensitization with horse gamma-globulin. It was also shown that either spleen or lymph node cells, but not serum, from OVA-fed donors transferred suppression to normal recipients. The transfer was mediated by antigen-specific suppressor T cells (Ts) that appeared to inhibit the induction phase (afferent limb) of the CMI response, since the Ts were only effective when transferred before or shortly after the onset of sensitization.     

Regulatory mechanism of delayed-type hypersensitivity in mice. II. Effect of suppressor cells on the development of memory cells for delayed-type hypersensitivity

Murine lymph node cells (LNC), which we showed previously to noncompetitively inhibit antibody-dependent cellular cytotoxicity (ADCC) to an erythrocyte target, were tested for their ability to inhibit ADCC to a tumor target, EL-4. Both a 4-hr ⁵¹Cr-release cytotoxicity assay and an overnight ¹²⁵IUdR (iododeoxyuridine) postlabeling cytostasis assay were used. Normal autologous lymph node cells inhibited spleen cell-mediated ADCC in both assays. Inhibition by LNC was dose dependent, but comparable numbers of sheep erythrocytes did not inhibit, indicating that LNC-mediated inhibition was not simply a matter of crowding. Inhibitory activity was enriched in LNC after removal of Fc receptor-bearing cells on EA monolayers.